We have published (Re et al. Neuron, 2014) that wild-type spinal motor neurons (MNs) are selectively killed by both mouse familial mutant SOD1 (mSOD1)-expressing astrocytes and human sporadic ALS astrocytes-or their conditioned medium (CM)-through a caspase-independent mechanism. In this study, we also show that necrostatin-1 (Nec-1), an allosteric inhibitor of the kinase function of the receptor interacting protein-1 (RIP1), which is an obligatory mediator of necroptosis, affords full protection against MN death in our in vitro models. Consistent with the RIP1 finding, our pilot data demonstrate that silencing RIP3 and inhibiting mixed lineage kinase domain-like (MLKL), two other known determinants of necroptosis, also provide MN protection. These findings offer the first experimental evidence that necroptosis regulates neuronal death in a model of chronic human neurological disorder. Yet, remarkably, aside from RIP1, RIP3, and MLKL, the molecular network of necroptosis, especially in neurons, remains elusive. Thus, as an initial phase toward unraveling the neuronal molecular network of necroptosis, we propose a 2 year scope of work consisting of two sequential steps: First, to assign the complex gene expression changes that occur during necroptosis to the actions of a limited number of regulatory genes, we will use: (i) our MN interactome (a cell type-specific regulatory network); and (ii) RNAseq data obtained from purified mouse embryonic stem cell-derived MNs exposed to CM made with either mSOD1-astrocytes (toxic condition) or wild-type SOD1 astrocytes (non-toxic control condition). So as to differentiate between general changes in MNs induced by astrocytes and those involved in necroptosis, each experiment will be performed in the presence or the absence of the antagonist Nec-1. Candidate master regulators (MRs; i.e. sets of transcription factor and signaling pathway genes causally associated with the phenotype) of necroptosis will be identified using the MARINa algorithm. Second, to validate the role of the candidate MRs-identified in Step 1-in the MN death phenotype, each will be targeted using shRNA strategies as shown in our pilot data. This validation will be done in both our in vitro mouse and human models by monitoring both MN survival and neurite length. Also to be performed in Step 2 are multiplex qRT-PCR experiments on MN samples, in each a MR will be individually knocked-down. The goal here is to examine whether any of the identified MRs regulate the expression of the others. As future studies, we propose: (1) to use the information generated herein to develop a genomic signature and new reagents/tools to study necroptosis in both post-mortem mouse models of and patients with neurodegenerative disorders such as, but not limited to ALS; and, (2) to assess the role of the most promising MRs in the expression of the disease phenotype in transgenic mouse model of ALS.